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The Noncanonical Roles of Two Primordial Molecules in Flagella

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The Noncanonical Roles of Two Primordial Molecules in Flagella Marquette University e-Publications@Marquette Dissertations (2009 -) Dissertations, Theses, and Professional Projects The oncN anonical Roles of Two Primordial Molecules in Flagella Xiaoyan Zhu Marquette University Recommended Citation Zhu, Xiaoyan, "The oncaN nonical Roles of Two Primordial Molecules in Flagella" (2017). Dissertations (2009 -). 734. http://epublications.marquette.edu/dissertations_mu/734 THE NONCANONICAL ROLES OF TWO PRIMORDIAL MOLECULES IN FLAGELLA by Xiaoyan Zhu, B.S. A Dissertation Submitted to the Faculty of the Graduate School, Marquette University, in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Milwaukee, Wisconsin August 2017 ABSTRACT THE NONCANONICAL ROLES OF TWO PRIMORDIAL MOLECULES IN FLAGELLA Xiaoyan Zhu, B.S. Marquette University, 2017 Motile cilia and flagella are ancient organelles that eukaryotic organisms today still rely on to thrive in their natural environment. Not surprisingly, accumulated evidence has shown that the intricate motility machinery, the microtubule-based axoneme, is evolutionarily conserved down to the molecular level. This notion is epitomized by the signature axonemal complex, the radial spoke (RS). The RS is part of a control center conferring the high frequency and tightly regulated movement. Key RS proteins discovered in biflagellate green alga, Chlamydomonas reinhardtii, are also generated by nearly all ciliated organisms, including Homo sapiens. Among them are two subunits from primordial protein families, nucleoside diphosphate kinase (NDK) and heat shock protein (HSP) 40, that are positioned at a critical juncture where two arms merge into a singular stalk in the Y-shaped complex. While it is well accepted that NDKs and HSP40s maintain nucleotide homeostasis and assist HSP70 chaperone in protein folding respectively, these actions fail to explain observations in myriads of vital cellular processes. Using the experimental approaches possible in the biflagellate green algae, genetics in particular, this dissertation discovers non-canonical applications of these two spoke proteins in the assembly and motility of the RS and entire flagellum. The versatility sheds light on the canonical mechanisms, the diverse processes adopting the non-canonical mechanisms, and their preservation since their conscription perhaps by the cell ancestral to all eukaryotes. i ACKNOWLEDGEMENTS Xiaoyan Zhu, B.S. My deepest gratitude goes to my mentor, Dr. Pinfen Yang, for her greatest patience in cultivating me to be a critical thinker and for her care and guidance of my growth in and outside of the laboratory. I feel blessed to work with a passionate scientist with fantastic mind and insight for the past six years. Her mentoring motivates me to be a better student, scientist and person every day. I would like to thank all my committee members, Dr. Kathleen Karrer, Dr. Rosemary Stuart, Dr. Allison Abbott and Dr. Martin St. Maurice, for their insightful suggestions, feedback and guidance. I also would like to thank our collaborators for their great support and beautiful works. I would like to thank the best lab member, Yi Liu, for all the help and critical evaluation of my work. I am also thankful to my best friends Pinqing Kan, Yan Li and Yulin Zhao, for their precious friendship and warm familial care. Most importantly, I would like to thank my parents and grandparents. It is their invaluable love and support that made my dream of studying aboard come true. Especially, I am thankful to my dearest Mother, my best lifetime friend, who has always being there for me during the stressful times. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS………………………………………………………………. i LIST OF TABLES………………………………………………………………………..vi LIST OF FIGURES………………………………………………………………….......vii LIST OF ABBREVIATIONS………………………………………………………...…..ix CHAPTER 1: INTRODUCTION 1.1 Cilia and Flagella……………………………………………………………...1 1.2 Chlamydomonas as a Flagella Model Organism……………………………..3 1.3 Intraflagellar Transport – A Tale of Two Cities………………………………4 1.4 The RS Complex………..……………………...…………….………………. 7 1.4.1 The Role of RS…………………………………………..………......8 1.4.2 The Composition of the RS………………………………………....9 1.4.3 The Assembly of the RS..…….………………………………...….11 1.4.4 The Two Primordial Molecules in the Neck of the RS………...…..13 1.4.4.1 HSP40………………………………………..…………..14 1.4.4.2 NDK……………………………………………..……….18 1.5 Objectives of This Dissertation……….………………………………...…....20 1.5.1 Elucidation of NDK5 in the RS …………………………………...21 1.5.2. Elucidation of the Noncanonical Role of Spoke HSP40……….…21 CHAPTER 2: MATERIALS AND METHODS 2.1 Materials……………………………………………………………………..22 2.1.1 Chlamydomonas Strains………………………………………..…..22 2.1.2 Culture Conditions…………………………………………...…….22 iii 2.1.3 Antibodies…………………..……………………………….….….22 2.2 Molecular Biology………………………………………………………...…26 2.2.1 PCR-based Genotyping………………………………………...…..26 2.2.1.1 Characterization of the Insertional Site in ndk5……….....26 2.2.1.2 Characterization of Mutation Site in pf33……………......26 2.2.2 Engineering of Genomic Constructs…………………………..…..26 2.2.2.1 Engineering of NDK5 Genomic DNA Constructs…….…26 2.2.2.2 Engineering of HSP40 Genomic DNA Constructs…..…..27 2.3 Chlamydomonas Experiments……………………………………………….30 2.3.1 Backcross……………………………………………………...…...30 2.3.2 Transformation……………………………………………...……...30 2.3.3 Flagellar Length Quantification……………………...…………….30 2.3.4 Dikaryon Rescue…………………………………...………………31 2.4 Biochemistry.………………………………………………………………...31 2.4.1 Flagella Preparation and Western Blot……….………………...….32 2.4.2 Purification of Bacterial Recombinant Proteins…………...………32 2.4.3 Pull-down Assay……………………………………………….......32 2.4.4 Nucleotide Diphosphate Kinase Activity Assay…………...………33 2.5 Microscopy…………………………………………………………………..33 2.5.1 Light Microscopy………………………………...………………...33 2.5.2 Electron Microscopy……………………………………...………..34 2.6 Modeling……………………………………………………………………..34 CHAPTER 3: GENERAL AND SPECIFIC PROMOTIONS OF FLAGELLAR ASSEMBLY BY A FLAGELLAR NUCLEOSIDE DIPHOSPHATE KINASE iv 3.1 Introduction…………………………………………………………………..36 3.2 Results………………………………………………………………………..39 3.2.1 NDK5H121A Minigene Caused Paralyzed Short Flagella……...…39 3.2.2 RS Defects in Paralyzed Flagella of an ndk5 Insertional Mutant….42 3.2.3 RS Deficiencies of ndk5 Flagella…………………………………..43 3.2.4 Short Flagella of ndk5………………………………………...……44 3.2.5 3D Reconstruction of NDK5 Axoneme with Cryo-electron Tomography……………………………………………………………...47 3.2.6 Full Rescue of ndk5 Phenotypes by NDK5 and NDK5H121A…...….48 3.2.7 Rescue of the Short Flagella of a Spoke-less pf14 Allelic Mutant……………………………………………………………...…….52 3.2.8 Dominant Negative Effect of NDK5H121A Genomic DNA……...…54 3.2.9 Hypophosphorylated NDK5 in the Cell Body………………….….57 3.3 Discussion……………………………………………………………………59 3.3.1 NDK5 is a Structural Protein for Proper Assembly of the RS Head- Neck Region……………………………………………...………………60 3.3.2 NDK5 Promotes Phosphorylation-related Assembly……...………60 CHAPTER 4: NON-CANONICAL ACTIONS OF HSP40 IN THE ASSEMBLY OF THE FLAGELLAR RS COMPLEX 4.1 Introduction……………………………………………………………..……65 4.2 Results………………………………………………………………………..68 4.2.1 Discovery of a HSP40 Transposon Insertional Mutant………...….68 4.2.2.Cryo-electron Tomography of HSP40-minus RSs………………...74 4.2.3 Identical Assembly Polarity but Distinct Efficiencies in the Repair of HSP40 and RSs……………………………………………....…………..76 v 4.2.4 Low Affinity of Recombinant HSP40 and NDK5………………....83 4.2.5 Structural Modeling of HSP40 and the RS Neck…………………..85 4.3 Discussion……………………………………………………………………89 4.3.1 HSP40 is Needed for RS Structural Stability……………………...89 4.3.2 Delayed Repair of Existing HSP40-minus RSs Sheds Light on HSP40 and Dikaryon Rescue……………………………………….……90 4.3.3 Modeling Predicts the Actions of HSP40……………………….....92 CHAPTER 5: DISCUSSION 5.1 New Spokeneck Model………………………………………………..……..95 5.2 Refined Structural Roles of NDK5…………………………………………100 5.3 Inter-subunit Crosstalk of NDK…………………………………………….101 5.3.1 Inter-subunit Crosstalk for the Canonical Mechanism of NDK….102 5.3.2 Implications of Inter-subunit Crosstalk.………………………….102 5.4 Structural Role of Spoke HSP40……………………………………………104 5.5 Trafficking and Assembly of Spoke HSP40………………………………..105 5.6 Potential Mechanisms of DN Phenotypes ………………………………….111 BIBLIOGRAPHY………………………………………………………..……………114 vi LIST OF TABLES Table 1-1. RS subunits in Chlamydomonas…………………………………..…………11 Table 1-2. Molecular domains in Group I and Group II human NDKs………………....20 Table 2-1. Strains generated in this dissertation………………………………………...23 Table 2-2. Antibodies used in this dissertation………………………………………….25 Table 2-3. Oligonucleotides used in this dissertation………………………………...…28 Table 3-1. List of strains described in this study and their corresponding motility, flagellar generation and RS assembly phenotypes……………………………………….52 Table 4-1. Chlamydomonas mutant strains deficient in the head or neck region of the RS………………………………………………………………...……………………....67 vii LIST OF FIGURES Figure 1-1. Chlamydomonas flagella……………………………………………………..2 Figure 1-2. Model of intraflagellar transport (IFT) dependent RS assembly…………….6 Figure 1-3. Surface rendering of RS in Chlamydomonas...................................................7 Figure 1-4. Generalized domain structures of HSP40 protein family………………...…15 Figure 1-5. Canonical model of the core HSP70-HSP40 machinery’s action in protein folding…………………………………………………………………………………....17 Figure 1-6. Crystal structure of a Group I NDK………………………………….……..19 Figure 3-1. NDK5H121A expressed from a minigene caused paralyzed short flagella in WT cells……………………………………………………………………………….....41
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